A major unresolved question in insect endocrinology is whether the nuclear receptor ultraspiracle (USP) has an active, ligand-binding function or, alternatively, is a non-liganded, even passive, heterodimer partner. Our previous NIH-funded protein biochemical studies emphasized screening of many point mutants to the ligand-binding domain of the Drosophila USP. The specific aims were to identify optimal mutant USPs for future testing of an in vivo USP ligand binding function of 2 transgenic fly assays. Our studies, supported by recent studies by others, were successful in demonstrating in cultured cell assays that the USP of Drosophila (flies) and Aedes (mosquitoes) can transduce transcriptional signaling by methyl epoxyfarnesoate, a natural juvenile hormone (JH) in flies and mosquitoes. The studies also show that this intracellular JH signaling requires a surface region on USP corresponding to a ligand-activated co-activator binding surface of the vertebrate counterpart, RXR. Using the cultured cell functional assay, we were successful in preparing point mutants to the USP ligand-binding pocket and co-activator binding surface that act as dominant negatives to block USP-dependent JH signaling in the cultured cells. On this basis, the recent NIH study section panel summary directed that our next proposal move past the protein biochemical studies by using "the mutants already in hand rather than looking for more mutants" and to now orient the studies "into a direct biological context". Therefore, in this new proposal, we take these Drosophila USP mutants (and corresponding Aedes USP mutants) will be used in the transgenic fly assays in order to ask the following questions: 1) Is the USP ligand-binding pocket needed for USP function in JH/ecdysone-driven larval-to-larval molting? 2) Does the USP ligand-binding pocket transduce JH effects on pupal-adult metamorphosis? These in vivo studies are the next logical step, building upon protein biochemical and cultured-cell assays, and are insect endocirinology's first direct in vivo tests of a ligand-binding function for any USP. The outcome will have broad implications for insect endocrinology, enabling specific tests on the Aedes USP, the results of which will yield new insights on hormonal signaling in the technically more difficult mosquito.